Research Project #3: Cell fate decision of liver tumor-initiating stem-like cells induced by alcohol. PROJECT SUMMARY/ABSTRACT Alcohol-associated hepatocellular carcinoma (HCC) is one of the leading causes of cancer deaths globally, yet efficacious therapeutic options are still limited. Central to therapy-resistance of HCC is the liver tumor-initiating stem-like cell (TIC) which we successfully isolated from HCC animal models and patients to understand their self-renewal mechanisms. We have identified a novel oncoprotein in TICs, TBC1D15 which facilitates phosphor-inactivation of the polarity protein NUMB by the TLR4-NANOG-AurkA-aPKC? pathway, leading to dissociation of p53 of NUMB, p53 ubiquitination and degradation, and TIC self-renewal. Functional significance of this mechanism is confirmed by marked reductions in liver tumor incidence and tumor-associated NANOG+ TICs by hepatocyte-specific expression of non-phosphorylatable NUMB or hepatocyte-specific deficiency of TBC1D15 in alcohol-fed HCV NS5A Tg mice. However, TBC1D15 expression endows non-transformed p53-deficient mouse hepatoblasts self-renewal activity. This suggests TBC1D15 has an unknown oncogenic mechanism besides promoting p53 loss. We have discovered TBC1D15 interacts with all NOTCH isoforms, activates the NOTCH pathway, and induces Nanog and TIC self-renewal in a NOTCH-dependent manner. Our data suggest TBC1D15 promotes NOTCH activation and antagonizes NUMB-mediated NICD destabilization. Two putative functional CSL/NICD sites in the Nanog gene were identified in a -5kb distal enhancer and a proximal promoter (-212/-151), both required for full Nanog transcription. We also mapped the TBC1D15-NICD interaction domain and identified a novel small molecule inhibitor which blocks the interaction and TBC1D15-dependent NICD-Hey1 and Nanog promoter activities. We hypothesize that TBC1D15 promotes oncogenesis by obligatory cooperation with the NOTCH pathway thus an inhibitor of TBC1D15-NICD interaction is therapeutic. We will pursue the following aims: Aim-1. Identify how TBC1D15 promotes the NOTCH pathway: Aim-1.1: Determine the role of TBC1D15-NOTCH interaction in NICD stabilization; Aim-1.2: Determine if TBC1D15 activates NOTCH. Aim-2. Identify how TBC1D15-activated NOTCH pathway contributes to TIC self-renewal and tumorigenesis: Aim-2.1: Determine how NICD supports Nanog transcription; Aim-2.2: Determine in vivo importance of the putative Nanog CSL/NICD sites in TIC-mediated tumorigenesis; Aim-2.3. Test the therapeutic efficacy of the novel inhibitor for the TBC1D15-NICD interaction in patient-derived xenograft (PDX) HCC model. Results from the proposed work will provide mechanistic insights into the novel oncogenic property of TBC1D15 and a pre-clinical basis for a future clinical trial using the novel inhibitor that antagonizes the interaction between TBC1D15 and NOTCH.